The antioxidant’s nightmare: Xanthine oxidoreductase

Ezine

Published: May 15, 2016

Author: Ryan De Vooght-Johnson

Channels: HPLC

Enzymes: essential for life

There are approximately 75,000 different enzymes in the human body. These critical proteins accelerate the reactions that break down food, allow nerve cells to communicate and muscles to contract – just to give a few examples.

One class of enzymes called oxidoreductases speeds up the transfer of electrons from one molecule to another. These enzymes are vital for metabolic processes, including aerobic and anaerobic respiration – the processes taking place in all living cells to produce energy.

Xanthine oxidoreductase (XOR) breaks down substances called purines (found in food and drink) into uric acid, which dissolves in the blood and travels to the kidneys, leaving the body in urine – a fundamental metabolic process. There are two types of this enzyme in the body however, and one can be harmful. One form of XOR produces free radicals: atoms, molecules or ions that have an unpaired electron. Free radicals are extremely reactive and can cause damage to fats, proteins and DNA in cells.

This dangerous form of XOR produces oxygen free radicals (also called reactive oxygen species), which cause cellular destruction and have been linked to ageing. The enzyme has therefore been associated with a wide range of disease states, including obesity, diabetes, heart disease, high blood pressure and kidney disease.

In order to understand how XOR causes these diseases, scientists need to measure its activity in the body. A team of Japanese scientists attempted this earlier this year, and achieved success with liquid chromatography/high-resolution mass spectrometry (LC-HRMS). They even reported the enzyme’s activity in mouse liver, kidney and blood samples.

Unfortunately, the method was not sensitive enough to measure XOR activity in other important samples, such as neurons, or human blood samples (which surprisingly have less XOR activity than mice).

A second attempt

Not satisfied with these results, the researchers decided to try again, publishing their latest results in the Journal of Labelled Compounds and Radiopharmaceuticals. This method again assesses the activity of XOR by measuring how much uric acid it produces. This time though, instead of using HR MS they used triple quadrupole MS (TQMS) – a more sensitive technique.

However, even this more sensitive technique had issues. TQMS was unable to distinguish the 15N2 isotope of uric acid from other isotopes with the same mass/charge ratio. To overcome this problem, the researchers measured the uric acid present in a small amount of human plasma using TQMS. Based on the results, they decided to use a different isotope of uric acid (13C2, 15N2) as the target analyte.

This was much more successful. Evaluating the standard calibration curve of the isotope of uric acid, they found good linearity within a wide concentration range of 20 to 4000 nM. Furthermore, the lowest concentration at which the analyte could be accurately detected was an impressive 33 times less than that of the previous HRMS-based method.

Mouse models man

To mimic human blood, the researchers diluted mouse plasma by over 1000 times and measured its XOR activity. They measured activity at around 4.4 pmol/100 µl/h – a level similar to healthy human blood.

Proven to detect XOR activity at a similar level to humans, this method could be used in both animal and clinical studies to accelerate research into clinically important disease states, including diabetes and heart disease.